Regulatory Spine Research Articles

ProKinO: A unified resource for mining the cancer kinome

The massive and exponential growth of genomic, structural, biochemical and clinical data on the human kinome poses major challenges in using existing data for cancer and clinical research. Here we describe an ontological framework for integrating and conceptualizing diverse forms of protein kinase data in a machine readable, human understandable form. We demonstrate the utility of this framework in mining the cancer kinome and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify novel mutational hotspots in key regulatory regions of the kinase domain, and demonstrate the impact of identified mutations on kinase activity and drug sensitivity. We find that mutations mapping to the regulatory spine activate the kinase domain through distinct mechanisms and contribute to drug resistance. User‐friendly tools to mine cancer mutations in the context of naturally occurring variants, pathways, and drug sensitivity profiles will be discussed.Support or Funding InformationNIH RO1 GM 11409‐01

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.

Functional Role of Histidine in the Conserved His-x-Asp Motif in the Catalytic Core of Protein Kinases.

The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.

Integration of signaling in the kinome: Architecture and regulation of the αC Helix

Eukaryotic protein kinases have evolved to be highly regulated and dynamic molecular switches that are typically kept in an inactive state and then activated in response to extracellular signals. The hallmark signature of an active kinase is a hydrophobic spine called the regulatory (R) spine, which consists of four residues, two in the N-lobe and two in the C-lobe. RS1 is in the catalytic loop, RS2 is the Phe in the DFG motif, RS3 is at the C-terminus of the αC-Helix, and RS4 is at the beginning of β4. Assembly of the R-spine is typically facilitated by phosphorylation of the Activation Loop. The assembled R-spine brings together all of the functional motifs that are essential for transferring the phosphate from ATP to a tethered protein substrate. This includes the G-Loop, which anchors the ATP, the catalytic loop, the DFG motif fused to the Activation Loop, and the αC-Helix. We focus here on the properties of the αC-Helix showing 1) how residues communicate with different parts of the molecule, 2) how it is recruited to the active site as a consequence of assembling of the R-spine, and 3) how it is regulated by linkers/motifs/proteins that lie outside the conserved kinase core. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Identification of a Novel HIP1-ALK Fusion Variant in Non–Small-Cell Lung Cancer (NSCLC) and Discovery of ALK I1171 (I1171N/S) Mutations in Two ALK-Rearranged NSCLC Patients with Resistance to Alectinib

Huntingtin-interacting protein 1 (HIP1) has recently been identified as a new fusion partner fused to anaplastic lymphoma kinase (ALK) in non–small-cell lung cancer (NSCLC). To date, two variants of HIP1-ALK (H21; A20) and (H28; A20) have been identified in NSCLC. However, the response of patients with NSCLC harboring HIP1-ALK to ALK inhibitors and potential resistance mechanisms to such remain unknown. Here, we report a patient with NSCLC harboring a novel HIP1-ALK fusion variant (H30; A20). This patient and another patient with EML4-ALK variant 3a/b initially responded sequentially to crizotinib and then alectinib, a next-generation ALK inhibitor, but developed acquired resistance to alectinib with the presence of a mutation in amino acid residue 1171 (I1171N and I1171S respectively) located in the hydrophobic regulatory spine (R-spine) of the ALK kinase in both the cases as identified by a comprehensive next-generation sequencing-based assay performed on biopsies of new liver metastases that developed during alectinib treatment.

Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues.

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Author SummaryEukaryotic protein kinases (EPKs) have a highly conserved enzymatic kinase core that is involved in the regulation of numerous cell signaling processes through the transfer of a phosphate group from adenosine triphosphate (ATP) to more than 30% of human proteins. EPKs have been implicated in numerous human diseases, including cancer, cardiovascular diseases, and diabetes, making them one of the most sought-after therapeutic drug targets. The lack of structural diversity of the active kinase core has created a bottle-neck for designing successful therapeutic inhibitors. Here we describe the intramolecular interactions required for differentiating between the active and inactive states of EPKs. Kinases contain a hydrophobic regulatory spine (“R-spine”) that is disassembled in inactive kinases, and here we define an additional hydrophobic “Shell” that surrounds one end of the R-spine. Biochemical analysis of the five nonconsecutive R-spine residues and three nonconsecutive Shell residues shows that proper assembly of the R-spine and Shell is essential for maintaining kinase activity. Structural analysis of the 172 known structures of EPKs without bound ligands led to the identification of four inactive conformations that correlate with the disassembly of the R-spine. Understanding the molecular elements involved in the regulation of kinase activity and the identification of these diverse groups of inactive conformations should aid the design of more specific therapeutic EPK inhibitors.

Confirming the Importance of the R-Spine: New Insights into Protein Kinase Regulation

Confirming the Importance of the R-Spine: New Insights into Protein Kinase Regulation

Locking the Active Conformation of c-Src Kinase through the Phosphorylation of the Activation Loop

Molecular dynamics umbrella sampling simulations are used to compare the relative stability of the active conformation of the catalytic domain of c-Src kinase while the tyrosine 416 in the activation loop (A-loop) is either unphosphorylated or phosphorylated. When the A-loop is unphosphorylated, there is considerable flexibility of the kinase. While the active conformation of the kinase is not forbidden and can be visited transiently, it is not the predominant state. This is consistent with the view that c-Src displays some catalytic activity even when the A-loop is unphosphorylated. In contrast, phosphorylation of the A-loop contributes to stabilize several structural features that are critical for catalysis, such as the hydrophobic regulatory spine, the HRD motif, and the electrostatic switch. In summary, the free-energy landscape calculations demonstrate that phosphorylation of tyrosine 416 in the A-loop essentially “locks” the kinase into its catalytically competent conformation.

Pseudokinases from a structural perspective

The catalytic (C) subunit of PKA was the first protein kinase structure to be solved, and it continues to serve as the prototype for the protein kinase superfamily. In contrast, by comparing many active and inactive kinases, we developed a novel 'spine' concept where every active kinase is composed of two hydrophobic spines anchored to a hydrophobic F-helix. The R-spine (regulatory spine) is dynamically assembled, typically by activation loop phosphorylation, whereas the C-spine (catalytic spine) is completed by the adenine ring of ATP. In the present paper, we show how the spine concept can be applied to B-Raf, specifically to engineer a kinase-dead pseudokinase. To achieve this, we mutated one of the C-spine residues in the N-lobe (N-terminal lobe), Ala481, to phenylalanine. This mutant cannot bind ATP and is thus kinase-dead, presumably because the phenylalanine ring fills the adenine-binding pocket. The C-spine is thus fused. However, the A481F mutant is still capable of binding wild-type B-Raf and wild-type C-Raf, and dimerization with a wild-type Raf leads to downstream activation of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK. The mutant requires dimerization, but is independent of Ras and does not require enzymatic activity. By distinguishing between catalytic and scaffold functions of B-Raf, we define kinases as being bifunctional and show that, at least in some cases, the scaffold function is sufficient for downstream signalling. Since this alanine residue is one of the most highly conserved residues in the kinome, we suggest that this may be a general strategy for engineering kinase-dead pseudokinases and exploring biological functions that are independent of catalysis.

“DFG-Flip” in the Insulin Receptor Kinase Is Facilitated by a Helical Intermediate State of the Activation Loop

We have characterized a large-scale inactive-to-active conformational change in the activation-loop of the insulin receptor kinase domain at the atomistic level via untargeted temperature-accelerated molecular dynamics (TAMD) and free-energy calculations using the string method. TAMD simulations consistently show folding of the A-loop into a helical conformation followed by unfolding to an active conformation, causing the highly conserved DFG-motif (Asp1150, Phe1151, and Gly1152) to switch from the inactive “D-out/F-in” to the nucleotide-binding-competent “D-in/F-out” conformation. The minimum free-energy path computed from the string method preserves these helical intermediates along the inactive-to-active path, and the thermodynamic free-energy differences are consistent with previous work on various other kinases. The mechanisms revealed by TAMD also suggest that the regulatory spine can be dynamically assembled/disassembled either by DFG-flip or by movement of the αC-helix. Together, these findings both broaden our understanding of kinase activation and point to intermediates as specific therapeutic targets.

Critical and synergy nodes in insulin-EGF signaling network

Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active catalytic subunits. PKA and all other protein kinase domains have a small amino-terminal lobe and large carboxyterminal lobe as determined by X-ray crystallography. The N-lobe and C-lobe form a cleft that serves as a docking site for MgATP. Nearly all active protein kinases contain a K/E/D/D signature sequence that plays important structural and catalytic roles. Protein kinases contain hydrophobic catalytic and regulatory spines and collateral shell residues that are required to assemble the active enzyme. There are two general kinds of conformational changes associated with most protein kinases. The first conformational change involves the formation of an intact regulatory spine to form an active enzyme. The second conformational change occurs in active kinases as they toggle between open and closed conformations during their catalytic cycles. Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. Imatinib was approved by the United States FDA for the treatment of chronic myelogenous leukemia in 2001; this small molecule inhibits the BCR-Abl protein kinase oncoprotein that results from the formation of the Philadelphia chromosome. More than two dozen other orally effective mechanism-based small molecule protein kinase inhibitors have been subsequently approved by the FDA. These drugs bind to the ATP-binding site of their target enzymes and extend into nearby hydrophobic pockets. Most of these protein kinase inhibitors prolong survival in cancer patients only weeks or months longer than standard cytotoxic therapies. In contrast, the clinical effectiveness of imatinib against chronic myelogenous leukemia is vastly superior to that of any other targeted protein kinase inhibitor with overall survival lasting a decade or more. However, the near universal and expected development of drug resistance in the treatment of neoplastic disorders requires new approaches to solve this therapeutic challenge. Cancer is the predominant indication for these drugs, but disease targets are increasing. For example, we can expect the approval of new drugs inhibiting other protein kinases in the treatment of illnesses such as hypertension, Parkinson's disease, and autoimmune diseases.

Controlling the Activity of the Tec Kinase Itk by Mutation of the Phenylalanine Gatekeeper Residue

The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

Identification of an Allosteric Signaling Network within Tec Family Kinases

The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the ‘regulatory spine’, which assembles and disassembles as a kinase switches between its active and inactive states, has been identified. Here, we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the Src homology 2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine, resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant, rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Bruton's tyrosine kinase R544 on the activation loop and Bruton's tyrosine kinase E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases.

Dynamics of PKA Signaling

The protein kinases represent one of the largest mammalian gene families and regulate in diverse and critical ways most biological processes in all eukaryotic cells. cAMP‐dependent protein kinase (PKA) has served as a prototype for understanding the structure and function of this protein kinase superfamily. The PKA catalytic subunit is assembled into an active conformation by phosphorylation of its activation loop where this single phosphate has profound effects on global structure and function. All active protein kinases share a common architecture that is built upon two hydrophobic spines comprised of residues from both the small (N‐) and large (C‐) lobes. These spines are anchored to the hydrophobic F Helix, which traverses the large helical C‐lobe. The regulatory spine is typically assembled by phosphorylation of the activation loop while the catalytic spine is completed by the adenine ring of ATP. The active kinase then opens and closes as it navigates through the catalytic cycle. The PKA C‐subunits are regulated by dimeric regulatory (R) subunits, which form an inactive tetrameric complex in the absence of cAMP. Catalytic activity is unleashed by cAMP binding with high affinity to the R‐subunits. Structures of R‐subunit monomers bound to the C‐subunit demonstrated that the R‐subunit undergoes major conformational changes as it releases cAMP and binds to the C‐subunit. The cooperative binding of cAMP then releases the C‐subunit. Specificity in PKA signaling is achieved in part by the isoform diversity of the regulatory subunits and by targeting of the holoenzymes to specific sites by A Kinase Anchoring Proteins (AKAPs). The AKAP docking site is localized to the N‐terminal dimerization/docking (D/D) domain of the R‐subunits, a four helix bundle that binds with high affinity to an amphipathic helix in the AKAP. Structures of the D/D domains from RIIα and RIα bound to the same AKAP peptide from D‐AKAP2 reveal an isoform‐specific code for binding to AKAPs. Crystallization of tetrameric holoenzymes, coupled with small angle Xray an neutron scattering, demonstrates how full length holoenzymes are assembled and anchored to AKAPs and reveals for the first time the full allosteric potential of PKA signaling.Support for this work was provided by NIH DK54441, GM19301, and GM34921.